Evaluation of the ability of commercial disinfectants to degrade free nucleic acid commonly targeted using molecular diagnostics.

BACKGROUND: Polymerase chain reaction (PCR) is an essential tool for rapid detection of pathogens, but is susceptible to cross-contamination by residual nucleic acid, leading to false-positive results. Adequate surface decontamination would help prevent this, but most protocols target infectious microbes rather than free nucleic acid. The aim of this study was to evaluate the ability of commercial surface disinfectants to degrade different representative classes of nucleic acid. METHODS: Commercial surface disinfectants with various active ingredients, as well as 10% chlorine bleach, were tested. Nucleic acid was dried on to stainless steel coupons and treated with disinfectant for 0-4 min prior to neutralization and quantification by quantitative reverse transcription PCR. The effective disinfectants were also evaluated in the presence of organic load. RESULTS: Only dilute chlorine bleach and the hypochlorite-based commercial disinfectant significantly degraded any type of free nucleic acid. Hydrogen-peroxide- and quaternary-ammonium-based disinfectants gave <1 log reduction after 4 min for all targets. Results were time-dependent for each target, which underscores the importance of adequate contact time. Organic load appeared to have little impact on the efficacy of hypochlorite-based disinfectants for nucleic acid degradation. CONCLUSIONS: This study demonstrates the importance of proper selection and application of disinfectant to remove residual nucleic acid when processing samples for molecular diagnostic testing.

Expression of K99 adhesion antigen controlled by the Escherichia coli tryptophan operon promoter.

The genetic determinant for the K99 adhesin of enterotoxigenic Escherichia coli B41 [O101:K99] has been cloned as a 7.0-kilobase BamHI-generated DNA fragment into the vector pBR322 by us and others (J. D. A. van Embden, F. K. de Graaf, L. M. Schouls, and J. S. Teppma, Infect. Immun. 29:1125-1133, 1980). Cells harboring one such construction, known as pK99-64, are capable of expressing K99 antigen on the cell surface. We replaced the natural promoter sequence for the gene encoding the K99 pilus subunit with a strong, inducible exogenous promoter, the E. coli tryptophan (trp) operon promoter, to construct the plasmid pBR-TrpK99. E. coli cells harboring pBR-TrpK99 or a similar construction in the plasmid pDR540, known as pKO-TrpK99, upon induction with 3-beta-indoleacrylic acid, produced about fourfold more K99 antigen than did cells bearing pK99-64 with the natural promoter. Expression of the pilus antigen was found to be under control of the tryptophan promoter. Plasmid instability was encountered, however, in cells bearing pKO-TrpK99 when the trp promoter was derepressed. Introduction of the aminoglycoside 3'-phosphotransferase gene of transposable element Tn5 into pKO-TrpK99 to generate pKON-TrpK99 effectively stabilized the plasmid in cells grown under identical conditions in medium containing kanamycin.

Efficient transformation of Serratia marcescens with pBR322 plasmid DNA.

Eight Serratia marcescens strains tested could be transformed with the plasmid pBR322. Transformants were selected on the basis of resistance to high levels of ampicillin (400 to 500 micrograms/ml). For six of the strains, the CaCl2- mediated transformation procedure developed for Escherichia coli was successful. For the other two strains, no transformants were obtained with the CaCl2-mediated transformation procedure unless the cells first received a heat treatment. Transformation frequency was dependent on DNA concentration, and no transformation was detected with linear pBR322 DNA. The stability and copy number of pBR322 were similar in S. marcescens and E. coli. As in E. coli, the pBR322 DNA was amplified in S. marcescens after inhibition of proteins synthesis. Based on these results, cloning in S. marcescens should be possible and pBR322 should be a useful cloning vehicle.